High resolution breakpoint junction mapping of proximally extended D4Z4 deletions in FSHD1 reveals evidence for a founder effect.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper arm muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting, molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally-extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and Southern blot strategy. Here, using next generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our results show that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis.

Chromosome 10q-linked FSHD identifies DUX4 as principal disease gene

BackgroundFacioscapulohumeral dystrophy (FSHD) is an inherited muscular dystrophy clinically characterised by muscle weakness starting with the facial and upper extremity muscles. A disease model has been developed that postulates that failure in somatic repression of the transcription factor DUX4 embedded in the D4Z4 repeat on chromosome 4q causes FSHD. However, due to the position of the D4Z4 repeat close to the telomere and the complex genetic and epigenetic aetiology of FSHD, there is ongoing debate about the transcriptional deregulation of closely linked genes and their involvement in FSHD.MethodDetailed genetic characterisation and gene expression analysis of patients with clinically confirmed FSHD and control individuals.ResultsIdentification of two FSHD families in which the disease is caused by repeat contraction and DUX4 expression from chromosome 10 due to a de novo D4Z4 repeat exchange between chromosomes 4 and 10. We show that the genetic lesion causal to FSHD in these families is physically separated from other candidate genes on chromosome 4. We demonstrate that muscle cell cultures from affected family members exhibit the characteristic molecular features of FSHD, including DUX4 and DUX4 target gene expression, without showing evidence for transcriptional deregulation of other chromosome 4-specific candidate genes.ConclusionThis study shows that in rare situations, FSHD can occur on chromosome 10 due to an interchromosomal rearrangement with the FSHD locus on chromosome 4q. These findings provide further evidence that DUX4 derepression is the dominant disease pathway for FSHD. Hence, therapeutic strategies should focus on DUX4 as the primary target.

CLIA Laboratory Testing for Facioscapulohumeral Dystrophy (FSHD): A Retrospective Analysis

Objective:To summarize facioscapulohumeral muscular dystrophy (FSHD) diagnostic testing results from the University of Iowa Molecular Pathology Laboratory.Methods:All FSHD tests performed in the diagnostic laboratory January 2015-July 2019 were retrospectively reviewed. Testing was by restriction enzyme digestion and Southern blot analysis with sequencing of SMCHD1, if indicated. Cases were classified as FSHD1 (4q35 EcoRI size ≤40kb; 1-10 D4Z4 repeats), FSHD2 (permissive 4q35A allele, D4Z4 hypomethylation, and pathogenic SMCHD1 variant), or non-FSHD1,2. We also noted cases with borderline EcoRI fragment size (41-43kb; 11 D4Z4 repeats), cases that meet criteria for both FSHD1 and FSHD2, somatic mosaicism, and cases with hybrid alleles that add complexity to test interpretation.Results:Of the 1594 FSHD tests included in the analysis, 703 (44.1%) were diagnosed with FSHD. Among these positive tests, 664 (94.5%) met criteria for FSHD1 and 39 (5.5%) met criteria for FSHD2. Of all 1594 cases, 20 (1.3%) had a 4q35 allele of borderline size, 23 (1.5%) were somatic mosaics, and 328 (20.9%) had undergone translocation events. Considering only cases with at least one 4q35A allele, D4Z4 repeat number differed significantly among groups: FSHD1 cases median of 6.0 (IQR: 4-7) repeats, FSHD2 cases 15.0 (IQR: 12-22) repeats, and non-FSHD1,2 cases 28.0 (IQR: 19-40) repeats.Conclusion:FSHD1 accounts for 94.5% of genetically confirmed cases of FSHD. The data show a continuum of D4Z4 repeat numbers with FSHD1 samples having the fewest, FSHD2 an intermediate number, and non-FSHD1,2 the most.

Characterizing the face in facioscapulohumeral muscular dystrophy

Objective To evaluate facial weakness in patients with FSHD to better define clinical signs, and pilot a facial weakness severity score. Methods 87 FSHD patients and 55 controls were video recorded while performing seven facial tasks. The videos were assessed by three independent examiners to compile an overview of signs of facial weakness. Next, videos were semi-quantitatively assessed using a newly developed 4-point facial weakness score (FWS). This score was evaluated and correlated to other FSHD disease characteristics. Results Patients had lower scores on the total FWS than controls (mean score 43 ± 28, range 4–118, vs 14 ± 9, range 0–35, p < 0.001) and on all seven individual facial tasks (all p < 0.001). 54% of patients had FWS scores outside the range of controls. Patients had more asymmetry between the left and right side of the face than controls. About 10% of the patients had very mild facial weakness. These were mostly males (89%) with longer D4Z4 repeat sizes of 7–9 units. More severe facial weakness correlated to more severe overall disease severity and shorter D4Z4 repeat size, but not to disease duration. Interobserver agreement for the FWS between three raters was low with a Fleiss Kappa of 0.437. Conclusion This study provides an overview of the clinical spectrum of facial weakness and its relation to other disease characteristics. The 4-point scale we introduced to grade the severity of facial weakness enables correlation of facial weakness to disease characteristics, but is not suited as clinical outcome measure for longitudinal studies.

Early-Onset Infantile Facioscapulohumeral Muscular Dystrophy: A Timely Review

Facioscapulohumeral muscular dystrophy (FSHD)—the worldwide third most common inherited muscular dystrophy caused by the heterozygous contraction of a 3.3 kb tandem repeat (D4Z4) on a chromosome with a 4q35 haplotype—is a progressive genetic myopathy with variable onset of symptoms, distribution of muscle weakness, and clinical severity. While much is known about the clinical course of adult FSHD, data on the early-onset infantile phenotype, especially on the progression of the disease, are relatively scarce. Contrary to the classical form, patients with infantile FSHD more often have a rapid decline in muscle wasting and systemic features with multiple extramuscular involvements. A rough correlation between the phenotypic severity of FSHD and the D4Z4 repeat size has been reported, and the majority of patients with infantile FSHD obtain a very short D4Z4 repeat length (one to three copies, EcoRI size 10–14 kb), in contrast to the classical, slowly progressive, form of FSHD (15–38 kb). With the increasing identifications of case reports and the advance in genetic diagnostics, recent studies have suggested that the infantile variant of FSHD is not a genetically separate entity but a part of the FSHD spectrum. Nevertheless, many questions about the clinical phenotype and natural history of infantile FSHD remain unanswered, limiting evidence-based clinical management. In this review, we summarize the updated research to gain insight into the clinical spectrum of infantile FSHD and raise views to improve recognition and understanding of its underlying pathomechanism, and further, to advance novel treatments and standard care methods.

Dnmt3b regulates DUX4 expression in a tissue-dependent manner in transgenic D4Z4 mice

Background Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown. Methods To determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation in Dnmt3b (Dnmt3bMommeD14 mice). Additionally, siRNA knockdowns of Dnmt3b were performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model. Results In mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown of Dnmt3b. In D4Z4-2.5/Dnmt3bMommeD14 mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3bMommeD14 mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3bMommeD14 mice or Smchd1MommeD1 mice. Only in D4Z4-2.5/Smchd1MommeD1 mice the immune cell populations were disturbed. Conclusions Our data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3bMommeD14 mice. In addition, the Smchd1MommeD1 variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD.

Homozygous nonsense variant in LRIF1 associated with facioscapulohumeral muscular dystrophy

ObjectiveFacioscapulohumeral muscular dystrophy (FSHD) is a heterogenetic disorder predominantly characterized by progressive facial and scapular muscle weakness. Patients with FSHD either have a contraction of the D4Z4 repeat on chromosome 4q35 or mutations in D4Z4 chromatin modifiers SMCHD1 and DNMT3B, both causing D4Z4 chromatin relaxation and inappropriate expression of the D4Z4-encoded DUX4 gene in skeletal muscle. In this study, we tested the hypothesis whether LRIF1, a known SMCHD1 protein interactor, is a disease gene for idiopathic FSHD2.MethodsClinical examination of a patient with idiopathic FSHD2 was combined with pathologic muscle biopsy examination and with genetic, epigenetic, and molecular studies.ResultsA homozygous LRIF1 mutation was identified in a patient with a clinical phenotype consistent with FSHD. This mutation resulted in the absence of the long isoform of LRIF1 protein, D4Z4 chromatin relaxation, and DUX4 and DUX4 target gene expression in myonuclei, all molecular and epigenetic hallmarks of FSHD. In concordance, LRIF1 was shown to bind to the D4Z4 repeat, and knockdown of the LRIF1 long isoform in muscle cells results in DUX4 and DUX4 target gene expression.ConclusionLRIF1 is a bona fide disease gene for FSHD2. This study further reinforces the unifying genetic mechanism, which postulates that FSHD is caused by D4Z4 chromatin relaxation, resulting in inappropriate DUX4 expression in skeletal muscle.

Type 1 FSHD with 6–10 Repeated Units: Factors Underlying Severity in Index Cases and Disease Penetrance in Their Relatives Attention

Molecular defects in type 1 facioscapulohumeral muscular dystrophy (FSHD) are caused by a heterozygous contraction of the D4Z4 repeat array from 1 to 10 repeat units (RUs) on 4q35. This study compared (1) the phenotype and severity of FSHD1 between patients carrying 6–8 vs. 9–10 RUs, (2) the amount of methylation in different D4Z4 regions between patients with FSHD1 with different clinical severity scores (CSS). This cross-sectional multicenter study was conducted to measure functional scales and for genetic analysis. Patients were classified into two categories according to RUs: Group 1, 6–8; Group 2, 9–10. Methylation analysis was performed in 27 patients. A total of 99 carriers of a contracted D4Z4 array were examined. No significant correlations between RUs and CSS (r = 0.04, p = 0.73) and any of the clinical outcome scales were observed between the two groups. Hypomethylation was significantly more pronounced in patients with high CSS (>3.5) than those with low CSS (<1.5) (in DR1 and 5P), indicating that the extent of hypomethylation might modulate disease severity. In Group 1, the disease severity is not strongly correlated with the allele size and is mostly correlated with the methylation of D4Z4 regions.

Does DNA Methylation Matter in FSHD?

Facioscapulohumeral muscular dystrophy (FSHD) has been associated with the genetic and epigenetic molecular features of the CpG-rich D4Z4 repeat tandem array at 4q35. Reduced DNA methylation of D4Z4 repeats is considered part of the FSHD mechanism and has been proposed as a reliable marker in the FSHD diagnostic procedure. We considered the assessment of D4Z4 DNA methylation status conducted on distinct cohorts using different methodologies. On the basis of the reported results we conclude that the percentage of DNA methylation detected at D4Z4 does not correlate with the disease status. Overall, data suggest that in the case of FSHD1, D4Z4 hypomethylation is a consequence of the chromatin structure present in the contracted allele, rather than a proxy of its function. Besides, CpG methylation at D4Z4 DNA is reduced in patients presenting diseases unrelated to muscle progressive wasting, like Bosma Arhinia and Microphthalmia syndrome, a developmental disorder, as well as ICF syndrome. Consistent with these observations, the analysis of epigenetic reprogramming at the D4Z4 locus in human embryonic and induced pluripotent stem cells indicate that other mechanisms, independent from the repeat number, are involved in the control of the epigenetic structure at D4Z4.

Intronic SMCHD1 variants in FSHD: testing the potential for CRISPR-Cas9 genome editing

BackgroundFacioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1.MethodsWe employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts.ResultsWe identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4.ConclusionsThe estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.